Modulation of induction properties of glucocorticoid receptor-agonist and -antagonist complexes by coactivators involves binding to receptors but is independent of ability of coactivators to augment transactivation

Coactivators such as TIF2 and SRC-1 modulate the positioning of the dose-response curve for agonist-bound glucocorticoid receptors (GRs) and the partial agonist activity of antiglucocorticoid complexes. These properties of coactivators differ from their initially defined activities of binding to, and increasing the total levels of transactivation by, agonist-bound steroid receptors. We now report that constructs of TIF2 and SRC-1 lacking the two activation domains (AD1 and AD2) have significantly less ability to increase transactivation but retain most of the activity for modulating the dose-response curve and partial agonist activity. Mammalian two-hybrid experiments show that the minimum TIF2 segment with modulatory activity (TIF2.4) does not interact with p300, CREB-binding protein, or PCAF, which also modulates GR activities. DRIP150 and DRIP205 have been implicated in coactivator actions but are unable to modulate GR activities. The absence of synergism by PCAF or DRIP150 with SRC-1 or TIF2, respectively, further suggests that these other factors are not involved. The ability of a TIF2.4 fragment (i.e. TIF2.37), which is not known to interact with proteins, to block the actions of TIF2.4 suggests that an unidentified binder mediates the modulatory activity of TIF2. Pull-down experiments with GST/TIF2.4 demonstrate a direct interaction of TIF2 with GR in a hormone-dependent fashion that requires the receptor interaction domains of TIF2 and is equally robust with agonists and most antiglucocorticoids. These observations, which are confirmed in mammalian two-hybrid assays, suggest that the capacity of coactivators such as TIF2 to modulate the partial agonist activity of antisteroids is mediated by the binding of coactivators to GR-antagonist complexes. In conclusion, the modulatory activity of coactivators with GR-agonist and -antagonist complexes is mechanistically distinct from the ability of coactivators to augment the total levels of transactivation and appears to involve the binding to both GR-steroid complexes and an unidentified TIF2-associated factor(s).     

Vitamin D-interacting protein 205 (DRIP205) coactivation of estrogen receptor alpha (ERalpha) involves multiple domains of both proteins

Vitamin D-interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17beta-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE(3)), DRIP205 coactivates ERalpha-mediated transactivation. DRIP205Delta587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205Delta587-636 also coactivates ERalpha. Moreover, both wild-type and variant DRIP205 also colocalize with ERalpha in the nuclei of transfected cells. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ERalpha and in interactions with ERalpha. Coactivation of ERalpha by DRIP205 does not require NR boxes, and variants with deletion of N-terminal (amino acids 1-639) and C-terminal (amino acids 576-1566) significantly coactivate ERalpha. DRIP205 resembles p160 coactivators that also interact with multiple regions of ERalpha; however, unlike p160 coactivators, DRIP205 coactivation of ERalpha does not require NR boxes.